There are two types of nuclear estrogen binding sites in normal and malignant tissues. Type I sites represent the classical estrogen receptor and nuclear Type II sites appear to mediate a specific nuclear response to estrogenic hormones. After estrogen administration, Type I receptor sites bind estradiol and this receptor-estrogen complex interacts with nuclear acceptor sites before the initiation of the transcriptional events that are associated with estrogen stimulation of tissue growth. In contrast, Type II sites bind estrogen with a higher capacity and a lower affinity than the classical estrogen receptor and do not appear to be translocated from the cytoplasm to the nucleus. Thus, although the levels of nuclear Type II sites are increased by estrogen administration, Type II sites remain in the cytoplasm after hormone administration. Nuclear Type II sites appear to mediate a specific nuclear response to estrogenic hormones and are highly correlated with uterine cellular hypertrophy and hyperplasia. Additionally, nuclear Type II sites are highly stimulated in malignant tissues such as mouse mammary tumors and human breast cancer. This observation is consistent with the findings that highly proliferative tissue has an increased number of nuclear Type II sites. Because the stimulation of nuclear Type II sites is closely correlated with the stimulation of uterine growth, it has been postulated that the Type II sites are the location for the mechanisms by which estrogens cause uterotropic stimulation. Furthermore, the presence of Type II sites on the nuclear matrix suggests a potential role in the regulation of DNA synthesis.
Nuclear Type II sites are constituents of many, if not all, non-malignant cells. Normally, Type II sites in non-malignant cells are occupied by methyl p-hydroxyphenyllactate (MeHPLA or methyl 3-(4-hydroxyphenyl)-2-hydroxypropionate). When MeHPLA binds to Type II sites, cell growth and proliferation of non-malignant tissues are slowed down or stopped. Conversely, malignant cells metabolize MeHPLA and thus there is insufficient binding to the nuclear Type II sites and the regulation of cell growth and proliferation is lost. Consequently all tumor cell populations examined have very high levels of unbound nuclear Type II sites. These sites should represent targets for the analogues of MeHPLA.
This invention discloses compounds which are not metabolized by malignant cells but which bind to nuclear Type II sites with high affinity. These compounds are very effective inhibitors of tumor cell proliferation and DNA synthesis. Since nuclear Type II sites have been observed in a variety of tumor cells, it is likely that analogues of MeHPLA and chemically related compounds will be effective inhibitors of a broad spectrum of tumors. Consequently, any tumor which contains nuclear Type II sites should respond to treatment with MeHPLA, its analogues, derivatives and chemically related compounds, including cancers of the pancreas, cervix, liver, brain, pituitary, prostate and other organ or tissue sites, as well as other cancers, such as leukemias, lymphomas, stromal myomas and leiomyomas, among others. Since MeHPLA will also block estrogen stimulation of normal cell growth such as that in the rat uterus (Table I), analogues of this compound may also be useful for the treatment of uterine hyperplasia, cervical hyperplasia, endometriosis and benign prostatic hypertrophy. Because non-proliferating non-malignant cells normally have their Type II sites bound with MeHPLA, the effects of the proposed compounds on non-malignant cell populations will be minimal to non-existent. For this reason, MeHPLA, its analogues, derivatives and chemically related compounds are also useful as prophylactic agents in the inhibition and prevention of cancer and non-malignant cell growth.
The precise physiological role of Type II sites is unknown, but inhibition of the nuclear Type II sites is associated with antagonism of uterotropic responses to estrogen. This is true for steroid antagonists such as dexamethasone, progesterone and triphenylethylene derivatives such as nafoxidine and clomiphene. While there is at least one endogenous inhibitor of estradiol binding to nuclear Type II sites, the specific inhibitor for the nuclear Type II sites has not been identified previous to this invention. Furthermore, the inhibitor of the present invention is specific to nuclear Type II sites and does not interfere with estradiol binding to cytoplasmic or nuclear Type I estrogen receptors.
The inhibitor is identified as methyl 3-(4-hydroxyphenyl)-2-hydroxypropionate and is an important regulator of cell growth and proliferation in normal and malignant tissues. The inhibitor is also known as methyl p-hydroxyphenyllactate or MeHPLA. These terms may be used interchangeably. Cell growth inhibition by this compound resides in its ability to interact with the high-affinity nuclear binding sites in normal and malignant cells which may be involved in the regulation of cellular proliferation and DNA synthesis. When MeHPLA is bound to nuclear Type II sites, cell growth and proliferation are inhibited. The endogenous 3-(4-hydroxyphenyl)-2-hydroxypropionic acid inhibits the cell growth much less effectively. This activity correlates with its low binding affinity for nuclear Type II sites.
An additional object of the present invention is a means for the prevention of cancer. Since MeHPLA is a normal constituent of mammalian cells, but metabolized by malignant cells, MeHPLA, structural analogues and chemically related compounds as described herein, which are not metabolized by tumors should be useful in the prevention of malignancy. These compounds should possess little if any side effects, and if taken in a low level maintenance dose should inhibit the proliferation of malignant cells.
Because MePHLA is such a potent inhibitor of cell growth, this compound, as well as its analogues and chemically related compounds were used as potential antitumor agents. The present invention discloses the potent antitumor activity of these compounds.